This invention relates to detection of antigens. More particularly, the invention relates to compositions and methods for detection of selected antigens in real time. In an illustrative embodiment, the invention relates to compositions and processes for sensitive detection of microbes and contaminants in complex samples, such as food samples, environmental samples, and the like, within about 30 minutes.
Bacterial spores are the most heat-stable form of microorganisms, are ubiquitous in the environment, and are therefore of great concern in food products, such as milk, that receive extensive heat treatments to prolong shelf life. Spore counts in milk from around the world vary from zero to greater than 22,000 cfu/ml depending on the climate of the region. S. A. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997). Bacillus stearothermophilus spores are one of the most heat-resistant bacterial spores and are found in high numbers in soil and water. Contaminating B. stearothermophilus spores survive extreme heat to germinate and grow at elevated product storage temperatures, which occur in foods transported in equatorial regions of the world.
While B. stearothermophilus is not commonly a problem, other bacilli often lead to food-borne illness or spoilage in a variety of foods. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus have all been implicated in outbreaks of food-borne illness and are commonly isolated from raw and heat treated milk. M.W. Griffiths, Foodborne Illness Caused by Bacillus spp. other than B. cereus and Their Importance to the Dairy Industry, 302 Int. Dairy Fed. Bulletin 3-6 (1995). B. cereus is also responsible for a sweet curdling defect in milk as well as being pathogenic. W.W. Overcast &K. Atmaram, The Role of Bacillus cereus in Sweet Curdling of Fluid Milk, 37 J. Milk Food Technol. 233-236 (1973). A mesophilic heat resistant bacillus similar to Bacillus badius, has been isolated from extreme temperature processed milk (D147=5 sec; P. Hammer et al., Pathogenicity Testing of Unknown Mesophilic Heat Resistant Bacilli from UHT-milk, 302 Int. Dairy Fed. Bulletin 56-57 (1995)). B. badius is a mesophilic organism and grows readily at room temperature, making it a likely candidate for spoiling temperature-processed foods. There have been 52 confirmed cases of B. badius in UHT milk in Europe and two cases outside of Europe. P. Hammer et al., supra. Lack of a rapid spore assay that can be used in milk contributes to the difficulty of prediction of post processing spoilage, thereby limiting the shelf life and product safety. H. Hofstra et al., Microbes in Food-processing Technology, 15 FEMS Microbiol. Rev. 175-183 (1994). Such an assay could be used in a hazard analysis critical control point (HACCP) plan allowing raw materials with high spore loads to be diverted to products that do not pose a food safety risk to consumers.
The standard method for quantifying spores in milk involves heat-shocking and an overnight plate count. G. H. Richardson, Standard Methods for the Examination of Dairy Products (1985). This is time-consuming and yields historical information. The food industry needs microbiological assays to yield predictive information for maximum benefit in HACCP analysis and risk assessment. An enzyme-linked immunosorbent assay (ELISA) capable of detecting greater than 106 cfu/ml of B. cereus spores in foods has been reported, but was unacceptable due to antibody cross-reactivity. Y. H. Chang & P. M. Foegeding, Biotin-avidin Enzyme-linked Immunosorbent Assay for Bacillus Spores in Buffer and Food, 2 J. Rapid Methods and Autom. Microbiol. 219-227 (1993).
Techniques to increase sensitivity of immunosorbent assays have focused on more efficient reporter labels, such as faster catalyzing reporter-enzymes; signal amplification, such as avidin- or streptavidin-biotin enzyme complexes; and better detectors, such as luminescence and fluorescence. L. J. Kricka, Selected Strategies for Improving Sensitivity and Reliability of Immunoassays, 40 Clin. Chem. 347-357 (1994); P. Patel, Rapid Analysis Techniques in Food Microbiology (1994). Immunomagnetic antigen capture is used extensively to separate and identify Escherichia coli and Salmonella from foods. C. Blackburn et al., Separation and Detection of Salmonellae Using Immunomagnetic Particles, 5 Biofouling 143-156 (1991); P. M. Fratamico et al., Rapid Isolation of Escherichia coli O157:H7 from Enrichment Cultures of Foods Using an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113 (1992); L. Krusell & N. Skovgaard, Evaluation of a New Semi-automated Screening Method for the Detection of Salmonella in Foods within 24 h, 20 Inter. J. Food Microbiol. 124-130 (1993); A. Lund et al., Rapid Isolation of K88+ Escherichia coli by Using Immunomagnetic Particles, 26 J. Clin. Microbiol. 2572-2575 (1988); L. P. Mansfeild & S. J. Forsythe, Immunomagnetic Separation as an Alternative to Enrichment Broths for Salmonella Detection, 16 Letters Appl. Microbiol. 122-125 (1993); A. J. G. Okrend et al., Isolation of Escherichia coli O157:H7 Using O157 Specific Antibody Coated Magnetic Beads, 55 J. Food Prot. 214-217 (1992); E. Skjerve & Olsvic, Immunomagnetic Separation of Salmonella from Foods, 14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright et al., Immunomagnetic Separation as a Sensitive Method for Isolating Escherichia coli O157 from Food Samples, 113 Epidemiol. Infect. 31-39 (1994). However, these methods involve either a preincubation or a subsequent incubation step (usually 18 to 24 hours) to increase the cell numbers for detection. Immunomagnetic capture greatly shortens E. coli and Salmonella testing, but long incubation times limit this method for predictive information. Immunocapture has also been used to quantitate Bacillus anthracis spores in soil samples using luminescent detection, J. G. Bruno & H. Yu, Immunomagnetic-electrochemiluminescent Detection of Bacillus anthracis Spores in Soil Matrices, 62 App. Environ. Microbiol. 3474-3476 (1996), but these efforts have led to tests that have a detection limit of 103 cfu/ml.
Considerable progress in the development of biosensors for microbial detection has been achieved in the last decade. These biosensors can be applied to medical, process control, and environmental fields. They must possess ideal features such as high specificity, simplicity, sensitivity, reliability, reproducibility, and speed. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as the recognition element for specific capture, numerous applications have been developed for detection of pathogenic bacteria. M. R. Blake & B. C. Weimer, Immunomagnetic Detection of Bacillus stearothermophilus Spores in Food and Environmental Samples, 63 J. Appl. Environ. Microbiol. 1643-1646 (1997); A. Burkowski, Rapid Detection of Bacterial Surface Proteins Using an Enzyme-linked Immunosorbent Assay System, 34 J. Biochem. Biophys. Methods 69-71 (1997); S. A. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods Using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L. A. Metherell et al., Rapid, Sensitive, Microbial Detection by Gene Amplification using Restriction Endonuclease Target Sequence, 11 Mol. Cell Probes 297-308 (1997); F. Roth et al., A New Multiantigen Immunoassay for the Quantification of IgG Antibodies to Capsular Polysaccharides of Streptococcus pneumoniae, 176 J. Inf. Dis. 526-529 (1997).
Methods for continuous flow immunoassay for rapid and sensitive detection of small molecules have been developed. For example, A. W. Kusterbeck et al., 135 J. Immunol. Methods 191-197 (1990), describes such a method in which detection of the antigen occurred within a matter of minutes. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the sample flow. Signal detection occurs downstream of the antigen recognition event.
In standard displacement flow immunoassays, the analyte of up to 1000 molecular weight in the sample creates an active dissociation of labeled antigens from an antigen binding site of an immobilized antibody, after which the labeled substance is measured downstream. W. A. Kaptein et al., On-line Flow Displacement Immunoassay for Fatty Acid-binding Protein, 217 J. Immunol. Methods 103-111 (1998), describes displacement in a flow system for detection of a small protein, cytoplasmic heart-type fatty acid-binding protein (15,000 molecular weight), a plasma marker for myocardial injury. This displacement system uses an inverse set-up: enzyme-labeled monoclonal antibodies are associated to immobilized antigen and are displaced by analyte in the sample.
F. Vianello et al., Continuous Flow Immunosensor for Atrazine Detection, 13 Biosens. Bioelectron. 45-53 (1998), describes detection of the hapten, atrazine, under continuous flow conditions using a micro-column containing immobilized monoclonal antibodies against atrazine and atrazine labeled with alkaline phosphatase. The equilibrium of the antibody-hapten system was achieved by continuous flow of the tracer (alkaline phosphatase-labeled atrazine) through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously downstream of the micro-column with an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine were added to the tracer flowing continuously through the micro-column, tracer bound to the antibody was displaced, with a consequent change in the detector signal.
C. H. Pollema & J. Ruzicka, Flow Injection Renewable Surface Immunoassay: A New Approach to Immunoanalysis with Fluorescence Detection, 66 Anal. Chem. 1825-1831 (1994), describes automatic heterogeneous immunoassays using a flow injection technique on a renewable surface. This assay relies on a minute amount of beads to form a reactive surface, which is interrogated by fluorescence spectrometry. Following the assay, the spent reactive surface is fluidically removed and replaced with a new layer of beads.
B. Mattiasson & M. P. Nandakumar, Binding Assays in Heterogeneous Media Using a Flow Injection System with an Expanded Micro-bed Adsorption Column, 8 Bioseparation 237-245 (1999), describes a competitive binding assay in a flow injection system wherein the adsorption step was carried out in an expanded bed column to increase the versatility of the assay an enable it to deal with samples containing particulate matter.
In view of the foregoing, it will be appreciated that compositions and methods for real time detection of selected antigens, such as contaminants in food and the environment, would be a significant advancement in the art.